Metabolomics experimental design

Question 1

How to measure the concentration of individual metabolites (eg T3)

Answer 1

ELISA test (Enzyme linked immunosorbent assay) A specific capture antibody is binded to the target antigen (ie T3) this is then linked to a enzyme labeled detection antibody which turns a non fluorescent substrate into a fluorescent substrate. You then get a signal that proportional to T3, using this you run a standard curve with known concentrations of T3

Question 2

Hypothyroid

Answer 2

low levels of T3

Question 3

Hyperthyroid

Answer 3

High levels of T3

Question 4

Problems with ELISA test

Answer 4

1. Only works for one specific metabolite
   2.

Question 5

What is metabolomics

Answer 5

Untargeted measurment and data analysis of the low molecular weight endogenous metabolites within a biological sample together these metabolites are refered to as the metabolome of a biological sample

Question 6

Relationship between the omics

Answer 6

Genome (what is possible) - Transcriptome ( First response) what gets activated from the genome - Proteome ( What makes it happen) the proteins doing the jobs - Metabolome (what happens ) the result that changes the cell- Phenotype

Question 7

Hypothesis driven research

Answer 7

Record comprehensive unbiased dataset on system of interest - “Mine” that dataset to determine effect on organism - Generate hypothesis - Record limited dataset specific to hypothesis - Determine if hypothesis is true or false

Question 8

Discovery driven untargeted metabolics assay

Answer 8

1. Relative quantitation (exact amount)
2. Global detection of a wide range of metabolites
3. we aquire data without prior knowledge of biologically relevant metabolites
4. HYPOTHESIS GENERATING
5. Metabolite identification required post data aquisition and prior to biological interpretation

Question 9

Hypothesis testing targeted metabolite assay

Answer 9

1. Quantification of a smaller number of metabolites (Typically less than 30)
2. Metabolite already known
3. Hypothesis testing
4. Quick route from data aquisition to biological knowledge

Question 10

PROS AND CONS of analytical startegies

Answer 10

Non targeted assay -
Holistic biological information
Targeted assay -
Easy metabolite identification
easy biological interpretation
Quantify easily

Question 11

Generic untargted metablomics work flow

Answer 11

Objective of study - experimental design - experiment - analytical experiment - Data integration/metabolite identification - biological interpretation

Question 12

How do we measure metabolites ?

Answer 12

Metabolites have very diverse physical and chemical properties
1. Mass spectrometry
2. NMR
3. electrochemical detection
4. IR spec
4. Raman spec

Question 13

Mass spec summary

Answer 13

Introduction of sample - ionizatioin - Mass seperation - Detection
m/z

Question 14

PCA

Answer 14

Techinque for examining the structure of complex data sets.